The long-term objective of the proposed studies is to elucidate the mechanism of mechanotransduction in bone. Our present bioengineering-oriented project developed a high-resolution piezoelectric mechanical loader and evaluated the role of mechanical stimulation in bone using cultured osteoblasts. The results reveal that (a) deformation of 3D collagen matrix can induce strain-induced fluid flow; (b) strain-induced fluid flow, and not strain itself, predominantly activates the stress-responsive genes in osteoblasts; and (c) architecture of 3D collagen matrix establishes a pattern of strain-induced fluid flow and molecular transport. Many lines of evidence in animal studies support enhancement of bone remodeling with strain of 1000 - 2000 microstrains. An unclear linkage between our in vitro studies and these animal studies is the role of strain and fluid flow in bone remodeling. In vitro osteoblast cultures including our current studies use 2D substrates or 3D matrices that hardly mimic the strain-induced fluid flow in vivo. This difference between in vitro and in vivo data makes it difficult to evaluate the role of strain and fluid flow in bone remodeling and anti-inflammation. First, microscopic strain in bone might be higher than the macroscopic strain measured with strain gauges. A local microscopic strain higher than 1000 - 2000 microstrains may therefore drive fluid flow in bone. Second, the lacunocanalicular network in bone could amplify strain-induced fluid flow in a loading-frequency dependent fashion. Lastly, interstitial fluid flow in bone might be induced by in situ strain as well as strain in a distant location, such that deformation of relatively soft epiphyses induces fluid flow in cortical bone in diaphyses. This renewal proposal will use mouse ulnae ex vivo as well as mouse in vivo loading to examine the above possible explanations for the data divergence. Specific aims include: (1) fabricating a piezoelectric mechanical loader for ex vivo and in vivo use; (2) quantifying ex vivo macroscopic and microscopic strains using electronic speckle pattern interferometry as well as molecular transport using fluorescence recovery after photobleaching; (3) conducting bone histomorphometry to evaluate ex vivo data; and (4) examining load-driven adverse effects with gene expression and enzyme activities (e.g., matrix metalloproteinases). Mechanical loads will be given in the ulna-loading (axial loading) and elbow-loading (lateral loading) modes. These two modes have been shown to enhance bone remodeling in the diaphysis with different patterns of strain distribution. Successful completion of the proposed renewal proposal will provide basic knowledge about induction of fluid flow in bone and establish a research platform for devising therapeutic strategies for strengthening bone and preventing bone loss.